Novel antagonistic yeast useful in controlling spoilage of agricultural produce methods of use thereof and compositions containg same

ABSTRACT

A biologically pure culture of a yeast of the species  Metschnikowia fructicola . The yeast is identified as NRRL Y-27328 and is capable of inhibiting growth of a deleterious micro-organism on a portion of a plant to which a biologically effective amount of a culture of the yeast is applied. Further disclosed is a composition for use in protection of agricultural produce including a biologically effective amount of  Metschnikowia fructicola  and a carrier. Further disclosed is an article of manufacture including packaging material and the disclosed composition which is identified for use in protection of agricultural produce from a deleterious micro-organism. Further disclosed is a method of inhibiting growth of deleterious micro-organism on a portion of a plant including applying at least one time an agriculturally effective amount of yeast of the genus Metschnikowia to the portion of a plant.

[0001] This application claims priority from U.S. Provisional PatentApplication 60/275,526 filed on Mar. 14, 2001.

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention relates to a novel antagonistic yeastuseful in controlling spoilage of agricultural produce, methods of usethereof and compositions containing same. Specifically, the presentinvention relates to the yeast Metschnikowia fructicola and to usethereof to inhibit growth of unwanted microorganisms on a portion of aplant, for example, foliage, flowers, fruit, roots or vegetables.

[0003] One of the most serious problems in the modern produce (fruit,vegetable and flower) industry is decay or spoilage of produce afterharvest. It is estimated that postharvest losses of fruits andvegetables are 50%. This loss is attributable to fungal and bacterialinfections to a great degree. In developing countries, postharvestlosses are often severe due to the lack of adequate handling andrefrigerated storage facilities. While developed nations have adequaterefrigeration, consumers in these countries often purchase produce whichhas been shipped great distances and stored for prolonged periods oftime. Postharvest decay of fruits and vegetables can be traced toinfections that occur either between flowering and fruit maturity orduring harvesting and subsequent handling and storage.

[0004] Synthetic fungicides such as imazalil and thiabendazole (TBZ)have traditionally been a primary means of controlling postharvestproduce loss. However, there is increasing global pressure to reduce theuse of toxic chemicals in the food industry. Consumers are concernedabout chemical residues on fruit and vegetables in general and areespecially uncomfortable with the idea of postharvest application ofchemicals.

[0005] In addition, environmentalists are becoming increasingly vocalabout chemical pesticide disposal and levels of chemical residues onfresh produce.

[0006] Concurrently, fungicide-resistant strains of pathogens haveemerged to most commonly used fungicides (e.g. TBZ, Imazalil, Rovral).Finally, some of the more effective fungicides such as Captan andBenlate have been deregistered and are no longer available. Further,postharvest treatment of some types of produce is not permitted. Inaddition, much postharvest spoilage is the result of pathogens whichcolonized produce prior to harvest.

[0007] All of these factors have contributed to increased interest inthe development of effective alternatives which pose no risk to humanhealth and the environment. Use of biological approaches such as naturalcompounds, induced resistance and antagonistic microorganisms in plantshave all been proposed as potential alternatives to synthetic fungicidesfor prevention or control of decay of agricultural produce.

[0008] Natural compounds are typically expensive to produce and oflimited efficacy. Of the biological approaches, they hold the leastpromise.

[0009] Induced resistance holds considerable promise in theory but hassuffered from two problems in other instances where it has been tried.Induced resistance based on genetically modified organisms (GMOs) hasoften had disappointing results because pathogens mutate when the GMOsare widely deployed in the field. However, the more complicated problemwith GMOs has proven to be resistance to GMOs in general by consumersand environmentalists.

[0010] This leaves development of antagonistic microorganisms as theremaining “acceptable” biological approach.

[0011] In recent years, research on the use of microbial biocontrolagents for the control of postharvest diseases of fruits has gainedconsiderable attention and has moved from the laboratory to commercialapplication. From these efforts, a large body of information regardingthe use of microbial biocontrol agent to control postharvest diseases isnow available (Droby et al., 2001). The selection of putative microbialantagonists has been based mainly on the ability of antagonists torapidly colonize fruit surfaces and wounds, out compete the pathogen fornutrients, and survive and develop under a wide range of temperatureconditions. Antagonists which can be used in the presence ofagricultural chemicals, including antibiotics, have not been previouslycharacterized.

[0012] A simple and reliable screening technique for selectingantagonists has been developed utilizing the wound site as a selectivemedium. Utilizing these procedures and other comparable protocols,several antagonistic bacteria, yeasts, and filamentous fungi have beenisolated and shown to protect a variety of harvested commoditiesincluding citrus and pome fruit against postharvest decay (Droby et al.,1989; Janisiewicz and Roitman, 1988; Chalutz and Wilson, 1990; Roberts,1990; Droby et al., 1991; Gullino et al., 1991; Janisiewicz, 1994; Lurieet al., 1995; Chand-Goyal and Spotts, 1996; El Ghaouth et al., 1998;Ippolito et al., 2000).

[0013] The success of some of these microbial antagonists in laboratoryand large scale studies has generated interest by several agro-chemicalcompanies in the development and promotion of postharvest biologicalproducts for control rots of fruits and vegetables. A number ofmicrobial antagonists have been patented and evaluated for commercialuse in postharvest treatment of produce. Currently, four antagonisticmicroorganisms, two yeasts, Candida oleophila, and Cryptococcus albidusand two strains of a bacterium, Pseudomonas syringae are commerciallyavailable under the trade names ASPIRE, YieldPlus, and BIOSAVE-110 andBIOSAVE-111 respectively.

[0014] Patents describing use of bacteria and yeasts for biologicalcontrol of fungal diseases of agricultural commodities include U.S. Pat.Nos. 5,314,691 (Coffey et al.); 5,270,059 (Janisiwicz et al.); 5,266,316(Elad et al.); 5,244,680 (Roberts); 5,238,690 (Elad et al.); 5,041,384(Wilson and Chalutz); 5,711,946 (Goyal and Roberts) and PCT publicationsWO 92/18009 (Shanmuganathan) and WO 91/01641 (Wilson et al.). Each ofthese prior art teachings is narrowly defined as a composition or methodcontaining/employing a disclosed species or strain of bacteria, fungusor yeast. None of these teachings includes a micro-organism which hasproved effective against a wide range of fungal pathogens in a widerange of agricultural commodities. Further, none of these patentscontain a hint or a suggestion that yeast of the genus Metschnikowia areuseful in preventing post harvest loss of produce.

[0015]Metschnikowia pulcherrima is known to have some efficacy inbiological control of a few deleterious micro-organisms on fruit(DeCurtis et al. (1996) Ann. Microbiol. Enzymol. 46: 45-55 and Piano etal. (1997) Postharvest Biol. Technol. 11:131-140), however it is aseparate and distinct species from Metschnikowia fructicola. Further,the apparently limited spectrum of antagonist activity of Metschnikowiapulcherrima renders it ill suited for use in the produce industry. Thusfar, only a limited spectrum of biocontrol activity for M. pulcherrimahas been demonstrated. This renders it ill suited to commercialprevention pre- and postharvest in a wide variety of agriculturalsettings.

[0016] There is thus a widely recognized need for, and it would behighly advantageous to have, a novel antagonistic yeast useful incontrolling spoilage of agricultural produce, methods of use thereof andcompositions containing same devoid of the above limitations.

SUMMARY OF THE INVENTION

[0017] According to one aspect of the present invention there isprovided A biologically pure culture of a yeast of the speciesMetschnikowia fructicola identified as NRRL Y-27328, the culture capableof competitively inhibiting growth of a deleterious micro-organism on afruit to which a biologically effective amount of the culture isapplied. Metschnikowia fructicola (MF), is referred to herein as strain#277 and has been deposited in the NRS culture collection (NRRL)National Center for Agricultural Utilization Research, Peoria, Ill., USAwhere it has been assigned deposit number NRRL Y-27328. This deposit hasbeen made in compliance with the terms of the Budapest Treaty.

[0018] According to another aspect of the present invention there isprovided a composition for use in protection of agricultural produceincludes, as an active ingredient, a biologically effective amount ofyeast of biologically pure Metschnikowia fructicola the compositionfurther containing a carrier.

[0019] According to yet another aspect of the present invention there isprovided a method of inhibiting growth of a deleterious micro-organismon a portion of a plant, the method includes applying at least one timean agriculturally effective amount of biologically pure culture of ayeast of the genus Metschnikowia to the portion of a plant. Applicationmay be pre-harvest, concurrent with harvest or post-harvest.

[0020] According to still another aspect of the present invention thereis provided an article of manufacture includes packaging material and acomposition identified for use in protection of agricultural producefrom a deleterious micro-organism includes, as an active ingredient, abiologically effective amount of yeast of biologically pureMetschnikowia fructicola the composition further containing a carrier.

[0021] According to further features in preferred embodiments of theinvention described below, there is provided a biologically pure strainof Metschnikowia fructicola having all of the identifyingcharacteristics of the biologically pure culture of NRRL Y-27328.

[0022] According to still further features in the described preferredembodiments there is provided a biologically pure mutant ofMetschnikowia fructicola, having all of the identifying characteristicsof the biologically pure culture of NRRL Y-27328.

[0023] According to still further features in the described preferredembodiments the deleterious micro-organism is selected from the groupconsisting of Botrytis cinerea, Aspergillus niger, Penicilliumdigitatum, Penicillium expansum, Rhizopus stolonifer Alternaria spp.,Molinilia spp. and Fusarium spp.

[0024] According to still further features in the described preferredembodiments the portion of a plant is selected from the group consistingof a stone fruit, a pome fruit, a citrus fruit, grapes, a vegetable, aflower bulb, an herb, a grain, a root, a leaf, a grain and berries.

[0025] According to still further features in the described preferredembodiments the yeast is supplied in a physiologic state selected fromthe group consisting of active and dormant.

[0026] According to still further features in the described preferredembodiments the yeast is supplied in a physical form selected from aliquid suspension, an emulsion, a powder, granules, a lyophylsate and agel.

[0027] According to still further features in the described preferredembodiments the composition further includes a chemical antibiotic.

[0028] According to still further features in the described preferredembodiments the chemical antibiotic is a fungicide or an antimicrobialagent or a pesticide.

[0029] According to still further features in the described preferredembodiments the fungicide includes at least one chemical selected fromthe group consisting of Iprodione, Thiabendazole, Imazalil(1-(2-2,4-Dichlorophenyl)-2(2-propenyloxy-ethyl)-1Himidazol),Fenhexamide, Pyrimethamil and a combination of Fludioxonyl andCyprodinil (e.g. Rovral, TBZ, Imazalil, Teldor, Mitos or Switch).

[0030] According to still further features in the described preferredembodiments the yeast of the genus Metschnikowia has all of theidentifying characteristics of the species Metschnikowia fructicolaidentified as NRRL Y-27328 or of any strain thereof or of any mutant ofsuch a strain.

[0031] According to still further features in the described preferredembodiments article of manufacture further includes an applicatordesigned and constructed to apply the yeast to the agricultural produce.

[0032] The present invention successfully addresses the shortcomings ofthe presently known configurations by providing a novel antagonisticyeast of the genus Metschnikowia useful in controlling spoilage of manytypes agricultural produce caused by a broad spectrum ofmicro-organisms. The present invention further provides methods of useof the yeast, compositions containing the yeast and articles ofmanufacture containing the compositions. The present invention isexpected to enjoy wide acceptance because it can provide post-harvestproduction from a pre-harvest application.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice. In the drawings:

[0034]FIG. 1 is a graph of cell number as a function of time for theyeasts Metschnikowia fructicola (Mf) of the present invention andMetshnikowia reukafii (231) and kluyveromyces thermotolerance (414)incubated at 4° C. indicating that Mf grows faster than other yeasts inthe cold.

[0035]FIG. 2 is a graph of colony forming units (cfu) per wound as afunction of time for the yeast Metschnikowia fructicola (Mf) of thepresent invention in citrus fruit surface wounds.

[0036]FIG. 3 is a histogram illustrating reduction of % decay caused byPenicillium digitatum (Pd) infection of red grapefruit by Metschnikowiafructicola (Mf) of the present invention and the effect of addition ofexogenous nutrients obtained from macerated grapefruit peel.

[0037]FIG. 4 is a histogram illustrating that response of green molddecay, caused by P. digitatum, on grapefruit to Metschnikowia fructicola(Mf) of the present invention is concentration dependent.

[0038]FIG. 5 is a photo of Petri dishes illustrating lack of longdistance inhibition of different postharvest pathogens by Metschnikowiafructicola (Mf) of the present invention in solid growth medium.

[0039]FIG. 6 is a histogram illustrating the correlation between ofchitinolytic activity of Metschnikowia fructicola (Mf) of the presentinvention and yeast isolates #47 and #273 with their biocontrol activityagainst Penicillium expansum on apples. The inset shows that the yeastMf produces high amounts of chitinases relative to the other yeastisolates.

[0040]FIG. 7 is a histogram illustrating the synergistic effect of theyeast Metschnikowia fructicola (Mf) of the present invention with thefungicide Rovral (Iprodione) against Botrytis cinerea of table grapes.

[0041]FIG. 8 is a graph of cfu as a function of time for Metschnikowiafructicola (Mf) of the present invention with various fungicides used tocontrol Botrytis cinerea and Penicilliun sp. Data presented indicatethat Mf is compatible/unaffected- with tested fungicides.

[0042]FIG. 9 is a graph of cfu/berry as a function of time forMetschnikowia fructicola (Mf) of the present invention, Metshnikowiareukafii (231) and kluyveromyces thermotolerance (414) on table grapes(cv. Superior) between field application and harvest.

[0043]FIG. 10 is a histogram comparing the effect of Metschnikowiafructicola (Mf) of the present invention to other yeast isolates ondecay of table grapes caused by Aspergillus niger.

[0044]FIG. 11 is a histogram comparing the effect of Metschnikowiafructicola (Mf) of the present invention to other yeast isolates ondecay caused by Botrytis of table grapes in a laboratory assay.

[0045]FIGS. 12 A and B are histograms of disease index and % decayedclusters respectively and the effect of Metschnikowia fructicola (Mf) ofthe present invention at 10⁸ cells/ml applied in the field as comparedto untreated controls and chemically treated grapes. Growth of pathogensAspergillus, Botrytis and Rhizopus is shown.

[0046]FIGS. 13 A and B are histograms of disease index and % decayedclusters respectively as a result of Rhizopus infection in the field andthe effect of Metschnikowia fructicola (Mf) of the present inventionrelative to M. raukaufili and K. thermotolerance.

[0047]FIG. 14 is a histogram showing decayed berries/3 Kg. for untreated(control) and grapes treated in the field with Metschnikowia fructicola(10⁸ cells/ml (MF) and 10⁷ cells/ml (MF⁻¹)) of the present invention.Decay was caused by Botrytis cinerea which developed postharvest.

[0048]FIG. 15 is a histogram showing decayed berries/Kg as a result ofstorage comparing untreated (control) and grapes treated withMetschnikowia fructicola (10⁸ (MF) cells/ml and 10⁷ (MF⁻¹) cells/ml) ofthe present invention. Decay was caused by Botrytis cinerea, Rhizopusstolonifer and Aspergillus niger.

[0049]FIG. 16 is a histogram comparing efficacy of the yeastMetschnikowia fructicola (Mf) of the present invention to Metschnikowiareukafii (231) and Kluyveromyces thermotolerance (414) and Mitos incontrolling damage to grapes in the field caused by Aspergillus andBotrytis and Rhizopus pathogens.

[0050]FIGS. 17A and B are histograms illustrating efficacy of the yeastMetschnikowia fructicola (Mf) of the present invention, as compared toC. oleophila and B. subtilis in controlling Fusarium oxysporum rot onEaster Lilly bulbs.

[0051]FIGS. 18 A and B are histograms illustrating efficacy of the yeastMetschnikowia fructicola (Mf) of the present invention, as compared toC. oleophila and B. subtilis in controlling Penicillium hirsutum rot onEaster Lilly bulbs.

[0052]FIG. 19 is a histogram illustrating total percent decay fromnatural infection of nectarines with various pathogens during storage at0 C for 30 days and additional 10 days of room temperature storage inthe presence of Metschnikowia fructicola (Mf) of the present inventionor Candida oleophila.

[0053]FIG. 20 is a histogram illustrating % infected wounds afterartificial infection of nectarines with Penicillium expansum in thepresence of Metschnikowia fructicola (Mf) of the present invention orCandida oleophila.

[0054]FIG. 21 is a histogram illustrating that the percentage ofinfected wounds from artificial infection of nectarines with Penicilliumexpansum decreases proportionally to the applied dose of Metschnikowiafructicola (Mf) of the present invention.

[0055]FIG. 22 is a histogram illustrating that total percent decay ofapples after artificial infection with Penicillium expansum varies withthe applied concentration of Metschnikowia fructicola (Mf) of thepresent invention.

[0056]FIGS. 23 A and B are a graph and a histogram respectively,illustrating the effect of pre-harvest application of Metschnikowiafructicola (Mf) of the present invention on the development of Botrytisrot of strawberries in the field (23A) and during post harvest storage(23B). Teldor 50% (WG power formulation)—015%; Teldor 500 (liquidformulation—0.15%; Switch—0.06% (60 g/dunam); Switch 0.1% (100 g/duman).

[0057]FIG. 24 is a series of histograms illustrating total % decay fromgreen mold decay in grapefruit in the presence or absence ofMetschnikowia fructicola (Mf) of the present invention or Metschnikowiaraukafii (231) or Candida oleophila (182).

[0058]FIG. 25 is a histogram showing concentration dependence of decay %of cherry tomatoes artificially infected with Botrytis cinerea toapplied Metschnikowia fructicola (Mf).

[0059]FIG. 26 is a histogram of % decayed fruits illustrating theability of Metschnikowia fructicola (Mf; 10⁸ cells/ml) of the presentinvention and of C. oleophila to reduce natural decay from (Botrytiscinerea) on sweet cherry during and after cold storage (30 days at 0° C.and after an additional 4 days of storage at 24° C.).

[0060]FIG. 27 is a histogram illustrating % decay from Botrytis cinerea(5×10⁴ spores/ml) on cherry tomato in the presence or absence ofMetschnikowia fructicola (Mf) of the present invention and at differentstorage temperatures.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present invention is of a new yeast species Metschnikowiafructicola which can be applied to agricultural produce to reduce pre-harvest and postharvest decay via competitive inhibition of a wide rangeof micro-organisms. Metschnikowia fructicola is referred to herein asstrain #277 and/or Mf and has been deposited in the NRS culturecollection (NRRL) National Center for Agricultural Utilization Research,Peoria, Ill., USA where it has been assigned deposit number NRRLY-27328. This deposit has been made in compliance with the terms of theBudapest Treaty.

[0062] Specifically, the present invention can be used to reduce theincidence and/or severity of fungal pathogens of grapes, citrus fruit,pome fruit, stone fruit, strawberries, flower bulbs, vegetables, roots,grains, foliage and herbs.

[0063] “Grapes”, as used in this specification and the accompanyingclaims, includes table grapes and wine grapes.

[0064] “Citrus fruit”, as used in this specification and theaccompanying claims, includes, but is not limited to, oranges,grapefruit, tangerines, clementines, lemons, limes, kumqwat, citroen,pomello, mandarin and hydrids derived therefrom.

[0065] “Pome fruit”, as used in this specification and the accompanyingclaims, includes, but is not limited to, apples, pears and quinces.

[0066] “Stone fruit”, as used in this specification and the accompanyingclaims, includes, but is not limited to, peaches, plums, nectarines,apricots, mangos.

[0067] For purposes of this specification and the accompanying claimsthe terms “inhibiting” and “inhibition” refer to retardation or delay ofa process. As such, inhibition may be deemed to occur if the processoccurs at a reduced rate as a result of application of a claimed yeast,a composition containing such a yeast, or as a result of practice of aclaimed method.

[0068] The invention is further of methods of use of the claimed yeast,compositions containing the claimed yeast and articles of manufactureincluding those compositions.

[0069] The principles and operation of protection of agriculturalproduce against unwanted decay via competitive inhibition according tothe present invention may be better understood with reference to thefigures and accompanying descriptions.

[0070] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement of thecomponents set forth in the following description or illustrated in thedrawings. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

[0071] According to one aspect of the present invention there isprovided A biologically pure culture of a yeast of the speciesMetschnikowia fructicola identified as NRRL Y-27328. This culture iscapable of competitively inhibiting growth of a wide range ofdeleterious micro-organisms on a portion of a plant to which abiologically effective amount of the culture is applied. Identifyingcharacteristics of Metschnikowia fructicola are set forth in“Metschnikowia fructicola, a New Ascoropic Yeast with Potential forBiocontrol of Postharvest Fruit Rots” (Kurtzman and Droby (2001) SystemAppl. Microbiol 24:in press) which is fully incorporated herein byreference. Briefly, NRRL Y-27328 differs from other members of the genusMetschnikowia in the D1/D2 domain of the 26S rDNA sequence.Specifically, NRRL Y-27328 differs by 2.2% from Metschnikowiapulcheirma, which is its closest known relative. It is well acceptedthat differences of 1% are sufficient for differentiation betweenspecies (Kurtzman and Robnett (1998) Antonie Leeuwenhoek 73:331-371).Therefore, Metschnikowia which differ from Mf by less than 1% in theD1/D2 domain of the 26S rDNA sequence are deemed to be within the scopeof the present invention.

[0072] Thus, any biologically pure strain of Metschnikowia fructicola,whether physically derived from the original deposit or independentlyisolated, is part of the present invention so long as it possesses allof the identifying characteristics of NRRL Y-27328. This includesbiologically pure mutants of Metschnikowia fructicola, so long as theyretain all of the identifying characteristics of NRRL Y-27328. Forpurposes of this specification and the accompanying claims, the term“mutant” includes both naturally occurring mutations and purposefulgenetic modifications such as introduction of point mutations, plasmids,phages, phagemids, cosmids and artificial chromosomes.

[0073] The deleterious micro-organism which Mf protects against include,but are not limited to, Botrytis cinerea, Aspergillus niger, Penicilliumdigitatum, Penicilium italicum, Penicillium expansum, Geotrichumcandidum, Rhizopus stolonifer, Alternaria spp., Molinilia spp, andFusarium spp.

[0074] The present invention is further embodied by a composition foruse in protection of agricultural produce. The composition includes, asan active ingredient, a biologically effective amount of yeast ofbiologically pure Metschnikowia fructicola. The composition furthercontains a carrier. As illustrated in examples set forth hereinbelow,Metschnikowia fructicola is biologically effective when delivered at aconcentration in excess of 10⁶ cells/ml, preferably in excess of 10⁷cells/ml, more preferably 10⁸ cells/ml, most preferably 10⁹ cells/ml ormore.

[0075] The yeast of the composition may be supplied in any physiologicstate such as active or dormant. Dormant yeast may be supplied, forexample, frozen (e.g in DMSO/glycerol), dried or lyophilized. Further,the yeast of the composition may be supplied in any physical formincluding, but not limited to a liquid suspension, an emulsion, apowder, granules, a lyophylisate or a gel.

[0076] The composition may be applied as spray or drench or as anaerosolized powder or ointment. If the composition includes dormantyeast, they may require re-activation prior to use, for example byrehydration and or incubation in a nutrient medium. Preferably, dormantyeast will become active when applied or subsequent to application.

[0077] In order to increase the overall efficacy of the composition, achemical antibiotic may be further included. Preferably, the chemicalantibiotic is a compatible fungicide, for example Iprodione (e.g.Rovral) or Thiabendazole (e.g. Apl-Luster, Arbotect, Mertect, Mycozol,TBZ, Tecto, and Thibenzole), Imazalil (i.e.1-(2-2,4-Dichlorophenyl)-2(2-propenyloxy -ethyl)-1Himidazol; e.g.Bromazil, Deccozil, Fungaflor, Freshgard, or Fungazil), Fenhexamide(e.g. Teldor), Pyrimethamil (e.g. Mitos) or a combination of Fludioxonyland Cyprodinil (e.g. Switch) or a chemical equivalent thereof or acombination including same. Alternately, or additionally, the chemicalantibiotic includes an antimicrobial agent or a pesticide.

[0078] The invention is further embodied by a method of inhibitinggrowth of a deleterious micro-organism on a portion of a plant, themethod includes applying at least one time an agriculturally effectiveamount of a biologically pure culture of a yeast of the genusMetschnikowia to the portion of a plant. Preferably, the yeast of thegenus Meischnikowia has all of the identifying characteristics of thespecies Metschnikowia fructicola identified as NRRL Y-27328 or of anystrain thereof or of any mutant of such a strain.

[0079] The invention is further embodied by an article of manufacturewhich includes packaging material and a composition identified for usein protection of agricultural produce from a deleterious micro-organism.The article of manufacture includes, as an active ingredient, abiologically effective amount of yeast of biologically pureMetschnikowia fructicola and further contains a carrier. Preferably, thearticle of manufacture further includes an applicator designed andconstructed to apply the yeast to the agricultural produce. As used inthis specification and the accompanying claims, the term “carrier”refers to any substance or diluent that does not cause significantirritation to agricultural produce or plants and does not abrogate thebiological activity and properties of the administered activeingredient. As such, the term specifically includes, but is not limitedto, aqueous solutions such as culture media, inert powders, and inertsolvents (e.g. water).

[0080] The claimed yeast species, compositions and articles ofmanufacture including same and methods of use thereof are expected tofind great utility in commercial agriculture. Their utility stems fromtheir broad spectrum of activity against important pathogens and fromthe wide range of plants/fruits to which they may be efficaciouslyapplied. In addition, Mf may be applied in the field, or concurrent withharvest, or during storage. Further, as demonstrated in examples hereinbelow, Mf is useful under a wide variety of storage conditions. Thus,the present invention allows preharvest application of a benign yeast asa means of preventing post-harvest decay of agricultural produce.

[0081] Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

[0082] Reference is now made to the following examples, which togetherwith the above descriptions, illustrate the invention in a non limitingfashion.

[0083] Generally, the nomenclature used herein and the laboratoryprocedures utilized in the present invention include molecular,biochemical, microbiological and recombinant DNA techniques. Suchtechniques are thoroughly explained in the literature. See, for example,“Molecular Cloning: A laboratory Manual” Sambrook et al., (1989);“Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M.,ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”,John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Immobilized Cells and Enzymes” IRL Press, (1986); “A PracticalGuide to Molecular Cloning” Perbal, B., (1984) and “Methods inEnzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide ToMethods And Applications”, Academic Press, San Diego, Calif. (1990);Marshak et al., “Strategies for Protein Purification andCharacterization—A Laboratory Course Manual” CSHL Press (1996); all ofwhich are incorpotaed by reference as if fully set forth herein. Othergeneral references are provided throughout this document. The procedurestherein are believed to be well known in the art and are provided forthe convenience of the reader. All the information contained therein isincorporated herein by reference.

Example 1 Isolation of Metschnikowia fructicola

[0084] The novel yeast species Metschnikowia fructicola was isolatedfrom the surface of grape berries (cv. Superior) grown in the centralpart of Israel. At various stages, individual berries were submersed insterile distilled water in 100 ml beakers and shaken vigorously for 2hours on rotary shaker at 120 rpm. Aliquots of 100 μl were removed fromthe wash liquid and plated on PDA (Potato Dextrose Agar; DIFCOLaboratories, U.S.A.) medium. Following 4-5 days of incubation, yeastcolonies were picked randomly according to colony characteristics (colorand morphology) and streaked individually on fresh medium to obtainbiologically pure cultures. Cultures were further purified by repeatedstreaking on PDA. Identification and characterization of the new specieswas done at the Microbial Genomics and Bioprocessing center, USDA-ARS,Peoria, Ill., USA. Metschnikowia fructicola was deposited at the NRRLunder the number Y-27328.

Example 2 Propagation of Metschnikowia fructicola

[0085]Metschnikowia fructicola is propagated under aerobic conditions attemperatures ranging from 5° C. to 37° C. Optimal growth temperature is20-27° C. The growth is in liquid medium (nutrient broth; Droby et al.,1989) with a neutral pH. The cell density of the yeast reaches itsmaximum (stationary stage) growth in 24-48 hours. For laboratory andsmall scale tests growth in Erlenmeyer flasks containing the medium andshaken on a rotary shaker is suitable. For large scale and commercialtests, fermentation tanks and industrial growth media are preferred. Theyeast cells are harvested by centrifugation using conventionallaboratory or industrial centrifuges. One ordinarily skilled in the artof fermentation culture will be able to scale up culture volumes usingsuitable growth media and commercially available equipment.

Example 3 Laboratory Assay of Activity of Metschnikowia fructicola onGrapes and Cherry Tomato

[0086] Individual grapes or cherry tomatoes were removed from clusters.Surface disinfection was accomplished by dipping for 1 min in 1% (v/v)sodium hypochlorite (pH 11.5). Disinfected fruit was mounted on maskingtape strips glued to PVC pads within an incubation box. The fruit waspunctured with a pin to a depth of 2 mm and 10 μl of an antagonist(Metschnikowia fructicola or as indicated) cell suspension were pippetedonto the wound site and left to dry for 1-2 hours. Fruit was theninoculated with 10 μl of conidial suspension of an appropriate fungalpathogen (B. cinerea or as indicated). Conidial suspensions wereobtained from one-week-old pathogen cultures incubated at roomtemperatures. Spore concentration was adjusted to 1-5×10⁴ conidia/ml.Each treatment was applied to three replicates of 7-10 individual fruit.Following the treatment, wet filter paper was placed in the incubationboxes which were covered with polyethylene to maintain high relativehumidity. The percentage of decayed berries/fruits in each replicate wasevaluated after 4-5 days at 20 C. This assay was employed in examples16, 17, 26 and 28 described hereinbelow.

Example 4 Field Test of Activity of Metschnikowia fructicola on Grape

[0087] The efficacy of Metschnikowia fructicola and other yeastantagonists against bunch rot of wine and table grapes was evaluated onvarious varieties of grapes in vineyards located in the northern,central and southern coastal plains of Israel. Thompson Seedless and‘Superior Seedless’ (table grapes) and ‘Sauvignon blanc’ (wine grapes)were tested. Experimental plots consisted of one to seven vines pertreatment in the different experiments, arranged as randomized blockswith at least four replicates. The yeast antagonist and chemicalstandards (Mitos 0.25%) were applied weekly up to 4 times until run-off,with hand driven back-sprayer.

[0088] The incidence of decay (% decayed bunches) in the grapeexperiments in the field was determined on the day of harvest (fieldevaluation). Forty clusters were sampled from each plot and scoredaccording to the causal agent of the decay and percentage of rot. Fortable grapes, the rot was also evaluated in storage. Approximately 3-5Kg of grapes were harvested from each plot and packed in plastic bagswhich were wrapped in polyethylene bags to create high relativehumidity. Rot development was evaluated after 3-4 weeks of storage at 0°C. followed by 4-7 days at 20° C. (cold storage ) or for 10 days at 20(shelf life). SO₂ standard treatment was used as a comparison. Datasummarized in FIGS. 12A and B indicates that Mf is comparable in itseffectiveness to the chemical antifungal.

Example 5 Metschnikowia fructicola Inhibits Decay of Sweet Cherries

[0089] The effect of the yeast antagonists Metschnikowia fructicola (MF)of the present invention and Candida oleophila (1-182) on thedevelopment of postharvest decay on sweet cherries were tested at aconcentration of 10⁸ cells/ml. Cherries were dipped for 1 min. in yeastcell suspensions, allowed to air dry and then stored under variousconditions: up to 6 days at 24° C.; 30 days at 0° C. followed by 4 daysat shelf life (room temperature); and 30 days at 0° C. under modifiedatmosphere (MA) packaging (P-Plus, Sidlaw Packaging, Bristol-UK)followed by 4 days of shelf life. The atmosphere in the bag used for MApackaging was determined by removing an aliquot of air and injecting itinto a GC gas chromatograph with a TDC detector and a Poropak column(Supelco, Bellepenta, Pa., USA). Number of decayed berries wasdetermined following the storage and shelf life period.

[0090] Data, summarized in FIG. 26, indicate that Mf is more effectivethan Candida oleophila.

Example 6 Growth Characteristics of Metschnikowia fructicola UnderRefrigeration

[0091] In order to demonstrate that Mf is useful under conditions ofcold storage, yeast cells were grown in Erlenmeyer flasks (50 mlmedia/100 ml flasks) containing liquid medium consisting of nutrientbroth, yeast extract and D-glucose (NYDB: Droby et al., 1989) on rotaryshakers placed in cold room at 4 C. Aliquots (1 ml) were asepticallywithdrawn from the growth medium at 1 day intervals, serially dilutedand plated on solid medium (NYDA: Droby et al., 1989). After 3-4 daysincubation at 25° C. the number of colonies growing on the medium werecounted and expressed as Log number of cells/ml. Data are summarized inFIG. 1. Mf grew much faster than M. raukaufii or K. thermotoleranceduring 6 days under refrigeration. This indicates that MF is more suitedto biological control of post harvest rot under cold storage conditionsthan M. reukaufii i or K. thermotolerance.

Example 7 Metschnikowia fructicola Colonizes Wounds in Citrus Fruit Peel

[0092] In order to demonstrate that Mf is capable of protecting woundsin a fruit surface, its ability to colonize and grow at the wound siteon a fruit surface was tested. The fruit was wiped with 95% ethanol andwounded (3×3 mm wounds) using a dissecting needle at four sites. Analiquot (20 μl) of the yeast cell suspension (2×10⁶ cells/ml) waspipetted into each wound. The fruit was incubated at 20° C. and atintervals of 1, 24 and 48 h, a 5×5 mm piece was cut from each of thefour wounds and vigorously shaken in 10 ml of sterilized distilled waterfor 1 h. Serial one-tenth dilutions of the washing liquid werte preparedand 100 μl of each dilution was plated on NYDA. The number of colonieswas counted during 48 h of incubation at 25° C. Each fruit containingfour wounds represented a single replicate, and each treatment contained6 replicates. Data (summarized in FIG. 2) are presented as cfu (colonyforming units) of Metschnikowia fructicola (Mf)/wound. Results indicatethat Mf is capable of replicating and colonizing a fruit surface woundat room temperature.

Example 8 Colonization by Metschnikowia fructicola Protects s Wounds inCitrus Fruit Peel by Competition for Nutrients

[0093] In order to establish that mf is capable of competing fornutrients surface sterilized grapefruits were wounded around the stemend, with three wounds per fruit. Each wound was made by inserting adissecting needle to a depth of 3 mm. Thirty μl of a water suspension ofthe antagonist Mf cells was pipetted into each wound. One to two hourslater, 20 μl of a spore suspension of P. digitatum (5×10⁴ spores/ml) wasapplied to each wound. Macerated grapefruit peel was prepared from watersoaked peel at the margins of P. digitatum induced lesions ongrapefruit. Ten grams of the peel was homogenized in blender, dilutedwith distilled water to 20% strength and then autoclaved. Aliquots ofconcentrated spore suspension of P. digitatum were added to the peelmacerate to give a final spore concentration of 5×10⁴ spores/ml, whichwas used to inoculate fruit wounds pretreated with a cell suspension ofMetschnikowia fructicola (Mf). Control fruits were inoculated with asimilar spore suspension of P. digitatum in water. Each fruit wasinoculated at three sites. Twelve fruits were used for each treatment togive a total of 36 inoculation sites per treatment. Percent woundinfection was measured after 5 days incubation at 24° C. Data aresummarized in FIG. 3. Results indicate that Metschnikowia fructicola(Mf) successfully protects against Penicillium digitatum (Pd) infectionof red grapefruit, but fails to do so in the presence of supplementarynutrients extracted from the peel. These results clearly indicate thatcompetition for nutrients plays a major role in Mf's protectivecapability.

Example 9 Metschnikowia fructicola Protects Against Green Mold Decay ina Concentration Dependent Manner

[0094] In order to demonstrate that the protective effect of Mf isconcentration dependent, surface sterilized grapefruits were woundedaround the stem end, as in Example 8. Thirty microliters of a watersuspension of the antagonist Mf cells was pipetted into each wound. Oneto two hours later, 20 ul of a spore suspension of P. digitatum (5×10⁴spores/ml) was applied to each wound. Percent of decay was determined 1week after incubation in humid conditions in plastic trays at 24 C.Twelve fruits per treatment were used. Data are summarized in FIG. 4.Results indicate that complete inhibition of decay was achieved at aconcentration of 10⁸ cfu with partial protection achieved at 10⁷ and 10⁶cfu.

Example 10 Metschnikowia fructicola Does Not Produce Antibiotics

[0095] Because there is great concern about widespread use ofantibiotics, the mechanism by which mf protects fruit was examined. Inorder to determine if the ability of Mf to protect fruit is mediated byantibiotic production, Mf was screened against three major fruitpathogens (Aspergillus niger, Botrytis cinerea, Penicillium digitatum).Yeast cells were streaked on one side of PDA plates. Agar plug (0.5×0.5mm) containing the fungal culture was placed on the other side of theplate and then plates were incubated at 25 C for one week.Representative results are presented in FIG. 5. No inhibition of thethree tested pathogens was observed. This demonstrates that Mf does notproduce diffusible antibiotics.

Example 11 Inhibition by Metschnikowia fructicola Correlates toChitinase Activity

[0096] To test relative biocontrol efficacy of Mf and other yeasts,apples were washed with tap water, dried, uniform wounds of 4 mm depthand 2-3 mm in diameter were made on the side of each fruit. Each woundwas inoculated with 40 μl of yeast suspension at a concentration of 10⁸cells/ml. After 3 h, 20 μl of suspension containing 10⁴ spore/ml of P.expansum were added to each wound. Treated apples were stored at ambienttemperature and high relative humidity Percent wound decay wasdetermined 10 days after inoculation. The number of infected (decayed)woun eachds was determined. Each treatment consisted of 3 replicates of10 fruits. Each fruit was wounded at one location.

[0097] Results are summarized in FIG. 6. Detection of chitinase activity(insert) was performed as follows: Sodium dodecyl sulfate (SDS)-PAGE wasperformed at pH 8.9 by using a 15% (w/v) polyacrylamid gel containing0.01%(w/v) glycol chitin as substrate and 0.1% (w/v) SDS. The crudeenzyme preparation, obtained from the supernatant of the yeasts liquidcultures were boiled for 2 min in 10% (w/v) sucrose and 2% SDS in 125 mMTris-HCl (pH 6.7). Elecrophoresis was run at room temp. for 1.5 h at 20mA. After Elecrophoresis, the gel was incubated overnight at 37 C onrotating apparatus in 200 ml of 100 mM sodium acetate buffer (pH 5). Atthe end of incubation period the gel was stained with 0.01% (w/v)Calcoflour white M2R in 0.3 M Tris-HCl (pH 8.9) for 5 min, and destainedby incubating the gel in 100 ml of distilled water with a gentle shakingfor 2 h. Lytic zones were visualized by placing the gel on under UVlight. Because Calcoflour white M2R does not stain hydrolyzed chitin,black bands in the Mf lane indicate that the yeast Mf produces highquantities of chitinase compared with the other tested yeasts Resultspresented in FIG. 6 clearly indicate that the superior ability of Mf toprotect apples against Penicillium expansum is well correlated tochitinolytic activity. Other yeasts lacking this activity (isolates #47and #273) were less effective against Penicillium expansum. This dataindicate that chitinolytic activity, as well as competition, contributeto the protective effect of Mf.

Example 12 Effect of Temperature on Metschnikowia fructicola

[0098] The ability Mf to remain viable after prolonged exposure totemperatures ranging from 0 to 42° C. was tested on solid potatodextrose agar plates. Mf was lightly streaked on the surface of PDAplates and incubated at the indicated temperatures for 4 days and thenmoved to 25° C. for an additional 4 days. Growth was evaluated visually.Results are summarized in Table 1. TABLE 1 Growth of the yeastMetschnikowia fructicola (Mf) at different temperatures. Initial 4 dayIncubation Time (days) at 25° C. incubation at After initial incubationTemp. ° C. 4+ 4 3 0 − − +++ 5 + + +++ 10 ++ ++ +++ 20 +++ +++ +++ 28 ++++++ +++ 37 − − +++ 42 − − −

[0099] These results indicate that Mf remains viable after storage at0-5° C. and that Mf is able to withstand temperatures lower than 37° C.

Example 13 Metschnikowia fructicola Acts Synergistically With ChemicalFungicides

[0100] Individual grapes (cv. Superior; table grapes) were surfacedisinfected by dipping for 1 min in 1% (v/v) sodium hypochlorite (pH11.5) and mounted on masking tape strips glued to PVC pads within anincubation box. The grapes were punctured with a pin to a depth of 2 mmand 20 μl of an antagonist cell suspension (10⁷ cells/ml) or Rovralbrand of Iprodione (5 ppm) or both were pippeted onto the wound site andleft to dry for 1-2 hours. Grapes were then inoculated with 10 μl ofconidial suspension of B. cinerea obtained from one-week-old pathogencultures incubated at room temperatures. Spore concentration wasadjusted to 1-5×10⁴ conidia/ml. Each treatment was applied to threereplicates of 7-10 grapes. Following the treatment, wet filter paper wasplaced in the incubation boxeswhich were covered with polyethylene tomaintain high relative humidity. The percentage of decayed grapes ineach replicate was evaluated after 4-5 days at 20° C. Results,summarized in FIG. 7, indicate that there is significant synergy betweenRovral brand of Iprodione and Mf.

Example 14 Metschnikowia fructicola is Compatible With a Varirty ofAgrochemicals

[0101] In order to establish that Mf is suitable for combination with avariety of commonly employed chemical pesticides, ten percent strengthof liquid medium (NYDB) with various concentrations of commonagrochemicals added was inoculated with a cell suspension of Mf (25 mlof 10⁹ cell/ml) and incubated on a rotary shaker at room temperature.The following commercially available agrochemicals were employed:TBZ(0.02%); Teldor (0.015%); Mitos (0.025%); Rovral (0.02%); Switch(0.01%); and Imazalil (0.005%). Samples were aseptically withdrawn fromthe at 0 time, 24 h and 48 h of incubation. Samples were seriallydiluted and aliquots of 30 μl of the dilutions were plated on PDA.Plates were incubated at 25° C. for 3-4 days and colony forming units(CFU) were counted.

[0102] Results, summarized in FIG. 8, indicate that MF is not affectedby the tested agrochemicals, even fungicides except Switch (0.01%) andImazalil (0.005%) which slowed, but did not prevent, its growth

Example 15 Metschnikowia fructicola Survives Under Field Conditions

[0103] In order to demonstrate that that Mf is more suited for use inthe field than others yeast strains, grapes were collected fromexperimental plots designated to evaluate the efficacy of the yeastantagonist Mf in controlling pre and postharvest rots of table grapes inthe central coastal area of Israel (Truman). Weekly spraying of theyeasts (starting from 21/6) at concentration of 10⁸ cells/ml using 231(Metschnikowia reukafii), 414 (Kluyveromyces thermotolerance) and Mf wasconducted. Grapes were collected on the first spraying date, after theclusters had dried and thereafter before each spray.

[0104] At each collection, five grapes per plot were sampled asepticallyinto 150 ml sterile plastic cups containing 20 ml of water and shaken ona rotary shaker at 200 rpm for 1 h. After 1:10 serial dilutions, 20 ulof each dilution were plated in Petri dishes containing Basal yeast agarmedium (BYA) containing 20 g glucose, 1 g yeast extract, 10 g proteasepeptone and 15 g agar amended with 250 mg Penicillin G (to suppressgrowth of bacteria) in 1 liter of distilled water. The Petri dishes wereincubated at room temperature for 3-4 days, after which the number ofcolonies were counted. Yeast viability is expressed as CFU/berry.Results, summarized in FIG. 9, indicate that Metschnikowia fructicola(Mf) survives better in the field than Metshnikowia reukafii (231) orkluyveromyces thermotolerance (414) on table grapes (cv. Superior).

Example 16 Metschnikowia fructicola Inhibits Growth of Aspergillus nigerand Botrytis cinerea on Grapes

[0105] In order to show that Mf is effective against important grapepathogens, individual grapes were removed from clusters, surfacedisinfected by dipping for 1 min in 1% (v/v) sodium hypochlorite (pH11.5) and mounted on masking tape strips as described hereinabove. Thegrapes were punctured with a pin as described hereinabove and anantagonist cell suspension was applied to the wound site and left to dryas described hereinabove. 30 μl of cell suspension of each strain atconcentration of 10⁸ cell/ml was used. Grapes were then inoculated with10 μl of conidial suspension of Aspergillus niger (FIG. 10) or Botrytiscinerea (FIG. 11). Conidial suspensions prepared and spore concentrationwas adjusted as described hereinabove.

[0106] Each treatment was applied to three replicates of 7-10 grapes.Following the treatment, wet filter paper was placed in the boxes whichwere covered with polyethylene to maintain high relative humidity. Thepercentage of decayed grapes in each replicate was evaluated after 4-5days at 20° C.

[0107]FIGS. 10 and 11 clearly indicate that Mf is the most effective ofthe assayed yeast strains in controlling growth of Aspergillus niger andBotrytis cinerea on wounds of table grapes. In FIG. 11 A42 indicatesCandida guilliemondii, 495 indicates Debaryomyces hansenii and strains243 and 509 are unidentified yeasts.

Example 17 Metschnikowia fructicola Controlsbunch Rot of Grapes

[0108] In order to further demonstrate the superior qualities of Mf inthe field, the efficacy of Mf relative to a chemical fungicide spray(mitos) against bunch rot of wine and table grapes was evaluated onvarious varieties in vineyards located in the northern, central andsouthern coastal plains of Israel on Thompson Seedless and ‘SuperiorSeedless’ (table grapes) and ‘Sauvingnon blanc’ (wine grapes).

[0109] Experimental plots consisted of one to seven vines per treatmentin the different experiments, arranged as randomized blocks with atleast four replicates. The yeast antagonist and chemical controls wereapplied weekly 4 times until run-off, with a hand driven back-sprayer.The incidence of decay in the wine and table grape experiments wasdetermined on the day of harvest. Forty clusters were sampled from eachplot and scored according to the causal agent of the decay andpercentage of rot. In the table grapes experiments, the rot was alsoevaluated in storage. Approximately 3-5 Kg of grapes were harvested fromeach plot and packed in plastic bags which were wrapped in polyethylenebags to create high relative humidity. Rot development was evaluatedafter 3-4 weeks of storage at 0 C followed by 4-7 days at 20° C.

[0110]FIGS. 12 A and 12 B clearly show that Mf applied to wine grapes ata concentration of 10⁸ cells/ml was more effective than Mitos (0.25%) incontrolling bunch rot caused by Aspergillus, Botrytis and Rhizopus.

[0111]FIGS. 13 A and 13 B clearly show that preharvest application ofthe yeast MF at concentrations as low as 10⁷ cells/ml was more effectivein controlling Rhizopus rot developed on grape bunches in the filed thanM. raukaufii or K. thermotolerance.

[0112]FIG. 14 clearly shows that Mf applied to table grapes (Cv.Superior) . . . at concentrations of 10⁸ (Mf) and 10⁷ (Mf⁻¹)cells/mlreduced disease severity after cold storage.

[0113]FIG. 15 clearly shows that Mf applied to table grapes (Cv.Thompson) at concentrations of 10⁸ and 10⁷ cells/ml prior to harvest wassuperior to chemical treatment with one SO₂ saturated pad per carton incontrolling bunch rot caused by Aspergillus for up to 2 weeks of storageat 20° C.

[0114]FIG. 16 clearly shows that Mf is more effective than Metchnikowiareukafii (231), Kluyveromyces thermotolerance (414) or mitos incontrolling damage to grapes caused by Aspergillus+Botrytis+Rhizopuspathogens.

[0115] In summary, these results indicate that use of Mf as a fungalantagonist is as effective as commonly employed chemical fungicides forcontrol of pre and postharvest bunch rot in grapes.

Example 18 Metschnikowia fructicola Protects Flower Bulbs From a Varietyof Pathogens

[0116] Easter Lilly bulbs were injured at one site and dipped for 1 minin cell suspension of the yeast Metschnikowia fructicola (Mf), Candidaoleophila or Bacillus subtilis at concentration of 10⁸ cells/ml, allowedto dry for 2-3 h and inoculated with spore suspension of Fusariumoxysporum (10⁵ spores/ml; FIGS. 17A and B) or of Penicillium hirsutum(10⁵ spores/ml; FIGS. 18 A and B) by spraying the bulbs until run off.Bulbs sprayed with water were used as controls. Percent of infectedbulbs was determined following storage period of two weeks at 9 Cfollowed by 1 week of storage at 20 C.

[0117] Data summarized in FIGS. 17 A and B show that Mf is moreefficient in controlling Fusarium oxysporum rot on Easter Lilly bulbsthan B. subtilis. Data summarized in FIGS. 18 A and B show that Mf ismore efficient in controlling Penicillium hirsutum rot on Easter Lillybulbs than C. oleophila. In summary, Mf offers broader protection forlily bulbs than C. oleophila or B. subtilis.

Example 19 Metschnikowia fructicola Inhibits Natural Infection of StoneFruit

[0118] In order to evaluate the effect of the yeast antagonistsMetschnikowia fructicola (Mf) and Candida oleophila on development ofnatural decay of stone fruit, ‘Flavortop’ nectarines were employed.Fruit was dipped into a cell suspension (10⁸ cells.ml⁻¹) of either Mf orCandida oleophila. Fruit dipped in tap water (20° C.) served as anegative control. Treated fruit was stored at 0° C. for 30 days and thenheld for 10 days at 24° C. At the end of this second incubation, decayincidence was determined. In all experiments, each treatment includedthree replicates of 30 fruit each.

[0119] Results summarized in FIG. 19 indicate that Mf was more effectivethan Candida oleophila in preventing natural infection of nectarineswith Alternaria spp., Monilinia fructicola, R. Stolonifer, B. cinerea,and P. expansum.

Example 20 Metschnikowia fructicola Inhibits Penicillium Infection ofStone Fruit Wounds

[0120] Stone fruit (‘Flavortop’ nectarines and ‘Swelling’ peaches) waswounded with a dissecting needle (1-2 mm deep) and 30 μl of theyeasts-cell suspensions (10⁸ cells ml⁻¹) were applied into each wound.The yeast antagonists used were either C. oleophila or Metschnikowiafructicola (Mf). Treated fruit was allowed to air dry and theninoculated with 20 μl of Penicillium expansum spore suspension (10⁵spores.ml⁻¹) and kept in plastic trays at 24° C. under humid conditions.The percentage of infected wounds was determined 4 and 5 days afterinoculation. Results summarized in FIG. 20 indicate that Metschnikowiafructicola (Mf) was more effective than Candida oleophila in preventingwound colonization by Penicillium expansum.

Example 21 Metschnikowia fructicola Inhibition of Penicillium Infectionof Stone Fruit is Concentration Dependent

[0121] Stone fruit (‘Flavortop’ nectarines and ‘Swelling’ peaches) werewounded as described in example 20 and 30 μl of varying Mf suspensions(10⁷, 10⁸ and 10⁹ cells/ml) were applied into each wound. Treated fruitwere handled as in example 20 hereinabove. Results, summarized in FIG.21, indicate that 10⁹ cells/ml of Mf was completely effective incompetitively inhibiting wound colonization by Penicillium expansumwhile lower concentrations provided significant degrees of protection.

Example 22 Metschnikowia fructicola Inhibits Penicillium Infection ofPome Fruit

[0122] Golden delicious apples were wounded with a dissecting needle(1-2 mm deep) and 30 ul of varying Mf yeast-cell suspensions (10⁸ and10⁹ cells.l⁻¹) were applied into each wound. Treated fruits were allowedto air dry and were then inoculated with 20 μl of Penicillium expansumspore suspension (10⁵ spores.ml⁻¹) and stored as in examples 20 and 21hereinabove. The percentage of infected wounds was determined at 5 daysafter inoculation.

[0123] Results summarized in FIG. 22 indicate that in pome fruit, as instone fruit, 10⁹ cells/ml of Mf was completely effective incompetitively inhibiting wound colonization by Penicillium expansum andthat lower concentrations provided significant degrees of protection.

Example 23 Pre-Harvest Application of Metschnikowia fructicola InhibitsBotrytis in Strawberry

[0124] In order to demonstrate the practicality of pre-harvestapplication of a yeast antagonist, MF cell suspension at a concentrationof 10⁸ cells/ml was applied by back sprayer to strawberry plants in acommercial field at weekly intervals. The first application was given atthe time of flowering. The yeast treatment was compared with differentcommercial fungicides at rates indicated in FIG. 23. After the fourthapplication, the number of decayed fruits in the field was counted ineach treatment (FIG. 23A). In addition, fruit were harvested, stored at1° C. for 3 days followed by 3 days at 24° C. to determine thepercentage of decayed fruit (FIG. 23B). These results demonstrate thatMf applied in the field provide protection from rot in the field andduring post harvest storage. Mf treatment gave a protection levelcomparable to that of commercial pesticides.

Example 24 Metschnikowia fructicola Inhibits Green Mold in Grapefruit

[0125] In order to demonstrate the versatility of Mf in preventing postharvest decay, green mold decay was determined in grapefruit after 6,14, 18, 24 days of storage at 20° C. The experiment was done in a pilotscale citrus packing line which simulated commercial operation. At theentering station the fruit received an extensive wash with plain waterfollowed by drying under fans blowing warm air. Fruit were then drenchedwith an Mf, Metschnikowia raukafii (231) or Candida oleophila (182) cellsuspension (10⁸ cells/ml), dryed under warm air blowers, waxed andpacked in commercial cartons. The fruit was kept at 20° C. and checkedafter different periods of incubation for the presence of decay.

[0126] Results summarized in FIG. 24 clearly indicate that Metschnikowiafructicola (Mf) was more effective than Metschnikowia raukafii (231) orCandida oleophila (182) in preventing development of green mold decay ingrapefruit

Example 25 Metschnikowia fructicola Inhibits Botrytis on Tomatoes

[0127] In order to demonstrate the utility of Mf in protectingvegetables, as well as fruits, during storage an assay was conducted oncherry tomatoes. Details of the assay are given in Example 3. Results,summarized in FIG. 25, clearly indicate that concentrations of Mf as lowas 10⁶ inhibit development of Botrytis cinerea after artificialinfection of cherry tomatoes.

Example 26 Metschnikowia fructicola Inhibits Botrytis on Cherries

[0128] In order to examine the relative efficacy of Mf in protectingstone fruit, the ability of C. oleophila and MF to retard development ofpostharvest decay on sweet cherries was tested. Each yeast antagonistwas applied at a concentration of 10⁸ cells/ml. Cherries were dipped for1 min in various salt solutions or yeast cell suspensions, allowed toair dry and then stored for 30 days at 0° C. followed by 4 days at 24°C. Results, summarized in FIG. 26, clearly indicate Metschnikowiafructicola (Mf) is superior to C. Oleophila in retarding development ofnatural decay from Botrytis cinerea on sweet cherries both underrefigeration and subsequent storage at room temperature.

Example 27 Metschnikowia fructicola Inhibits Botrytis on Tomatoes UnderVarious Storage Conditions

[0129] In order to determine the efficacy of Mf in protecting Cherrytomatoes under various storage conditions, tomatoes were treated withyeast and inoculated with B. cinerea as described hereinabove (Example3). Tomatoes treated with water and inoculated with B. cinerea served ascontrol. After treatment tomatoes were divided into three groups. Onegroup was incubated at 5° C. for 14 days, the second group was incubatedat 10° C. for 14 days and the third group was incubated at 20° C. for 5days. Results, summarized in FIG. 27, clearly indicate that MF providessignificant protection under all conditions tested. Especiallyimpressive is the complete protection afforded tomatoes by Mf underrefrigeration.

[0130] Although the invention has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

[0131] All publications, patents and patent applications mentioned inthis specification are herein incorporated in their entirety byreference into the specification, to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

REFERENCES

[0132] Chalutz, E., and Wilson, C. L., 1990, Postharvest biocontrol ofgreen and blue moldand sour rot of citrus by Debaryomyces hansenii.Plant Dis. 74:134-137.

[0133] Droby, S., Chalutz, E.,Wilson, C. L., and Wisniewski, M. E.,1989,. Characterization of the biocontrol activity of Debaryomyceshansenii in the control of Penicillium digitatum on grapefruit. Can. J.Microbiol. 35: 794-800.

[0134] Droby, S., Wilson, C. L., Wisniewski, M., and El-Ghaouth, A.2001. Biologically based technology for the control of postharvestdiseases of fruits and vegetables. In: Microbial Food Contamination, C.L. Wilson and S. Droby (eds.). CRC Press, Boca Raton, Fla.

[0135] Gullino, M. L., Aloi, C., Palitto, M., Benzi, D., and Garibaldi,A., 1991, Attempts atbiocontrol of postharvest diseases of apple. Med.Fac: Landbouw. Rijksuiv. Gent, 56: 195.

[0136] Janisiewicz, W. and Roitman, J.,1988, Biological control of bluemold and gray mold on apple and pear with Pseudomonas cepacia.Phytopathol. 78:1697-1700.

[0137] Janisiewicz, W. J., Peterson, D. L., and Bors,, R., 1994, Controlof storage decay of apples with Sporobolomyces roseus. Plant Disease78:466-470.

[0138] Droby, S., Chalutz, E., and Wilson, C. L., 1991, Antagonisticmicroorganisms as biological control agents of postharvest diseases offruits and vegetables. Postharvest News and Information 2: 169-173.

[0139] Lurie, S., Droby, S., Chalupowicz, L., and Chalutz, E., 1995,Efficacy of Candida oleophila strain 182 in preventing Penicilliumexpansum infection of nectarine fruits. Phytoparasitica 23:231-234.

[0140] Roberts, R. G., 1990, Biological control of mucor rot of pear byCryptococcus laurentii, C. flavus, and C. albidus. Phytopathol. 80:1051.

[0141] Chand-Goyal, T., and Spots, R. A. 1996. Control of postharvestpear diseases using natural saprophytic yeast colonists and theircombination with low doses of thiabendazole. Postharv. Biol. Technol.7:51-64.

[0142] Ippolito, A., El Ghaouth, A., Wilson, C. L., and Wisniewski, M.2000. Control of postharvest decay of apple fruit by Aureobasidiumpullulans and induction of defense responses. Postharv. Biol. Technol.19:265-272.

[0143] El Ghaouth, A., Wilson, C. L., Wisniewski, M. 1998.Ultrastructural and cytochemical aspect of the biocontrol activity ofCandida saitoana in apple fruit. Phytopathol 88: 282-291.

What is claimed is:
 1. A biologically pure culture of a yeast of thespecies Metschnikowia fructicola identified as NRRL Y-27328, saidculture capable of competitively inhibiting growth of a deleteriousmicro-organism on a portion of a plant to which a biologically effectiveamount of the culture is applied.
 2. A biologically pure strain ofMetschnikowia fructicola having all of the identifying characteristicsof the biologically pure culture of claim
 1. 3. A biologically puremutant of Metschnikowia fructicola, having all of the identifyingcharacteristics of the biologically pure culture of claim
 1. 4. Theyeast of the species Metschnikowia fructicola of claim 1, wherein saiddeleterious micro-organism is selected from the group consisting ofBotrytis cinerea, Aspergillus niger, Penicillium digitatum, Penicilliumexpansum, Geotrichum candidum, Rhizopus stolonifer, Fusarium spp andMolinilia spp.
 5. The yeast of the species Metschnikowia fructicola ofclaim 1, wherein said portion of a plant is selected from the groupconsisting of a stone fruit, a pome fruit, a citrus fruit, a grape, aberry, a vegetable and an herb.
 6. A composition for use in protectionof agricultural produce comprising, as an active ingredient, abiologically effective amount of yeast of biologically pureMetschnikowia fructicola said composition further containing a carrier.7. The composition of claim 6, wherein said yeast is supplied in aphysiologic state selected from the group consiting of active anddormant.
 8. The composition of claim 6, wherein said yeast is suppliedin a physical form selected from a liquid suspension, an emulsion, apowder, granules, a lyophylsate and a gel.
 9. The composition of claim6, further comprising a chemical antibiotic.
 10. The composition ofclaim 9, wherein said chemical antibiotic is selected from the groupconsisting of a fungicide, an antimicrobial agent and a pesticide. 11.The composition of claim 9, wherein said fungicide includes at least onechemical selected from the group consisting of Iprodione, Thiabendazole,Imazalil (1-(2-2,4-Dichlorophenyl)-2(2-propenyloxy-ethyl)-1Himidazol),Fenhexamide, Pyrimethamil and a combination of Fludioxonyl andCyprodinil.
 12. A method of inhibiting growth of a deleteriousmicro-organism on a portion of a plant, the method comprising applyingat least one time an agriculturally effective amount of biologicallypure culture of a yeast of the genus Metschnikowia to the portion of aplant,
 13. The method of claim 12, wherein said yeast of the genusMetschnikowia has all of the identifying characteristics of the speciesMetschnikowia fructicola identified as NRRL Y-27328.
 14. The method ofclaim 13, wherein said yeast of the genus Metschnikowia comprises astrain of the Metschnikowia fructicola of claim 13 having all of theidentifying characteristics thereof.
 15. The method of claim 13, whereinsaid yeast of the genus Metschnikowia comprises a mutant ofMetschnikowia fructicola having all of the identifying characteristicsthereof.
 16. The method of claim 12, wherein the portion of a plant isselected from the group consisting of a pome fruit, a stone fruit, acitrus fruit, a grape variety, a vegetable and a flower bulb.
 17. Themethod of claim 12, wherein said deletyerious micr-organism is selectedfrom the group consisting of Botrytis cinerea, Aspergillus niger andRhizopus stolonifer.
 18. An article of manufacture comprising packagingmaterial and a composition identified for use in protection ofagricultural produce from a deleterious micro-organism comprising, as anactive ingredient, a biologically effective amount of biologically pureyeast of the species Metschnikowia fructicola said composition furthercontaining a carrier.
 19. The article of manufacture of claim 18,further comprising an applicator designed and constructed to apply saidyeast to the agricultural produce.
 20. The article of manufacture ofclaim 18, wherein the agricultural produce is selected from the groupconsisting of a pome fruit, a stone fruit, a citrus fruit, a grapevariety, a flower bulb and a vegetable.
 21. The article of manufactureof claim 18, wherein said deleterious micr-organism is selected from thegroup consisting of Botrytis cinerea, Aspergillus niger, Penicilliumdigitatum, Penicillium expansum, Geotrichum candidum and Rhizopusstolonifer.